Fermentation Optimization Of Corynebacterium Nitrilophilus Nhase And Its Application In Surface Modification Of Polyacrylonitrile Fibers

Nitrile hydratase (EC 4.2.1.84 , NHase) catalyzes the hydration of nitriles to corresponding amides in the nitriles metabolism. It has been successfully applied in production of industrial chemicals acrylamide and vitamine such as nicotinamide. It can degrade the nitriles-containing wastes, which reducing the harm to environment. It can also be used in the surface modification of polyacrylonitrile fibers to enhance the fiber wettability and dyeability. With expanded application of NHase in the industries related to chemical engineering, food, environmental protection and textile process, the research on NHase has gained more attention.In this study, the fermentation optimization of NHase producing strain C. nitrilophilus in shaking flasks was investigated, then the application of NHase in the surface modification of polyacrylonitrile fibers was studied. In addition, C. nitrilophilus NHase gene was cloned and successfully expressed in the E.coli BL21(DE3). The main results are listed as follows:(1)The seed cultured of 24 h was used for fermentation and NHase activity achieved 16.2 U/mL at the cultivation of 72 h. Single-factor experiments and Orthogonal experiment were used to optimize the culture medium and culture condition of C. nitrilophilus in shaking flasks, the optimized results were as follows : glucose 15 g/L,yeast extract 3 g/L,NH4Cl 1 g/L, KH2PO4 0.5 g/L, K2HPO4?3H2O 0.5 g/L, glutamic acid 1 g/L, MgSO4?7H2O 0.2 g/L, NaCl 2 g/L, CoCl2?6H2O 0.07 g/L, the initial pH of 7.5, the temperature of 37℃, the inoculum size of 7% and a medium content of 50 ml culture medium in 500 ml flask, the inducer urea (10 g/L) was added at the 12 h. The NHase activity reached 58.3 U/mL compared with the initial activity of 16.2 U/mL, which enhanced 2.6 times after optimization.(2)In order to improve wettablity and dyeability of polyacrylonitrile fibers, the conditions for NHase acted on polyacrylonitrile fibers were studied. The optimized results were as follows: polyacrylonitrile fibers were pretreated for 30 min (80℃) by 5% isopropyl alcohol, then washed absolutely with water; the fibers were treated at 30℃, pH 6.0, the liquor ratio of 1:40, 160 r/min, shaking for 6 h, with NHase added 11 U/mL, then washed with water absolutely, drying at 60℃.Through the treatment of NHase, the fibers'wettablity and dyeability inhanced 124% and 142% respectively.(3)Cutinase, with the activity of esterase, can degrade vinyl acetate which is the second monomer of polyacrylonitrile fibers. After treatment, the superhelix structure of fiber can be loosen, which enhanced the affinity of NHase to the fiber. In this way, the efficiency of NHase treated the polyacrylonitrile fibers can be raised. Compound enzyme with cutinase and NHase were used for surface modification of polyacrylonitrile fibers and the wettability and dyeability of fiber increased. The analysis of fiber chemical composition by FT-IR ATR showed the increased amount of caobonyl and analysis the surface morphology of fiber by SEM showed the good modification efficiency treated by compound enzyme.(4)In order to increase the production of NHase, C. nitrilophilus NHase gene was cloned and expressed into E.coli BL21(DE3), but the expression level was low and the enzyme activity was 7.2 U/mL. So that increasing the expression level remained to be further study.